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Nipple aspirate fluid (NAF), a noninvasive liquid biopsy from at-risk women, contains nucleic acid and protein biomarkers from adjacent cancer cells, yet cfDNA use in NAF for breast cancer (BrC) screening remains insufficiently explored. A proof-of-concept study applied cfDNA bisulfite sequencing (cfBS) to NAF, detecting distinct tumor DNA methylation signatures and EMT-associated epigenetic changes that distinguished tumors from healthy controls and were supported by validation in TCGA.",{"@graph":69,"@context":123},[70,84,106],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/cell-free-dna-bisulfite-sequencing-reveals-epithelialmesenchymal-transition-signatures-for-breast-cancer/373697/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/cell-free-dna-bisulfite-sequencing-reveals-epithelialmesenchymal-transition-signatures-for-breast-cancer/373697.png","ImageObject",300,407,{"name":92,"@type":93},"Elsa","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-11-01","2026-09-23",true,{"@type":102,"interactionType":103,"userInteractionCount":105},"InteractionCounter",{"@type":104},"ViewAction",11,{"@type":107,"mainEntity":108},"FAQPage",[109,115,119],{"name":110,"@type":111,"acceptedAnswer":112},"What role does cfDNA play in breast cancer detection?","Question",{"text":113,"@type":114},"cfDNA released by malignant tumor cells provides epigenetic information that can indicate cancer status.","Answer",{"name":116,"@type":111,"acceptedAnswer":117},"Why is nipple aspirate fluid (NAF) relevant to cfDNA bisulfite sequencing?",{"text":118,"@type":114},"NAF is a noninvasive liquid biopsy that contains biomarkers from nearby cancer cells, offering a potentially enriched source for tumor DNA.",{"name":120,"@type":111,"acceptedAnswer":121},"How did the study link methylation patterns to epithelial–mesenchymal transition (EMT)?",{"text":122,"@type":114},"Differentially methylated genes were enriched for EMT, and a novel EMT scoring metric showed a more mesenchymal phenotype in breast cancer samples, validated using TCGA.","https://schema.org",{"og:url":83,"og:type":125,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":127,"canonical":83},"index,follow",{"doc_id":129,"site_id":62},373697,1790472562,{"code":4,"msg":5,"data":132},{"doc_id":129,"user_id":133,"nickname":92,"user_avatar":134,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":135,"file_id":136,"file_url":137,"file_type":138,"file_size":139,"view_count":105,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":56,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":145},137455077381,"https://ap-avatar.wpscdn.com/davatar_994ba38a5ba835b3df7d355c54d3ed8d","Article  \nCell-Free DNA Bisulfite Sequencing Reveals  \nEpithelial–Mesenchymal Transition Signatures for Breast Cancer  \nMinsun Stacey Jeon 1,2, Zehuan Ding 1,3, Casey Pei 2, Jing Li 1,2, Linglin Xie 3, Edward Sauter 4, * and Ke Kurt Zhang 1,2,3, *  \nAcademic Editor: Elena Bonanno  \nReceived: 5 August 2025  \nRevised: 29 August 2025  \nAccepted: 5 September 2025  \nPublished: 7 September 2025  \nCitation: Jeon, M.S.; Ding, Z.; Pei, C.; Li, J.; Xie, L.; Sauter, E.; Zhang, K.K. Cell-Free DNA Bisulfite Sequencing Reveals Epithelial–Mesenchymal Transition Signatures for Breast Cancer. Int. J. Mol. Sci. 2025, 26, 8723. [https://](https://)[ ](https://)[doi.org/10.3390/ijms26178723](doi.org/10.3390/ijms26178723)  \nCorrection Statement: This article has been republished with a minor change. The change does not affect the scientific content of the article and further details are available within the backmatter of the website version of this article.  \nCopyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ([https://creativecommons.org/](https://creativecommons.org/)[ ](https://creativecommons.org/)[licenses/by/4.0/](licenses/by/4.0/)) .  \n1 Center for Epigenetics and Disease Prevention, Institute of Biosciences & Technology, Texas A&M Health Science Center, Houston, TX 77030, USA; [minsunjeon@tamu.edu](minsunjeon@tamu.edu) (M.S.J.); [zehuan.d@tamu.edu](zehuan.d@tamu.edu) (Z.D.); [jing.li@tamu.edu](jing.li@tamu.edu) (J.L.)  \n2 Rigor and Reproducibility Core, Institute of Biosciences & Technology, Texas A&M Health Science Center, Houston, TX 77030, USA; [caseypei@tamu.edu](caseypei@tamu.edu)  \n3 Department of Nutrition, Texas A&M University, College Station, TX 77843, USA; [linglin.xie@ag.tamu.edu](linglin.xie@ag.tamu.edu)  \n4 National Cancer Institute, Division of Cancer Prevention, Rockville, MD 20850, USA  \n* Correspondence: [edward.sauter@nih.gov](edward.sauter@nih.gov) (E.S.); [kzhang@tamu.edu](kzhang@tamu.edu) (K.K.Z.)  \nAbstract  \nCell-free DNA (cfDNA), shed by malignant tumor cells into extracellular fluid, provides valuable epigenetic information indicative of cancer status. Nipple aspirate fluid (NAF), anoninvasive liquid biopsy from at-risk women, contains nucleic acid and protein biomarkers from adjacent cancer cells, showing promise for breast cancer (BrC) detection. However, despite its potential, the application of cfDNA in NAF for BrC screening is still underexplored. Here, we report a proof-of-concept study for using cfDNA bisulfite sequencing (cfBS) to assess tumor DNA methylation signatures from NAF samples. For four healthy individuals and three BrC patients, cfBS achieved greater than 20 × sequencing depth with an average coverage of 26.5 × on the genome. A total of 7471 differentially methylated regions were identified, with significant hypermethylation in BrC samples compared to healthy controls. Gene set enrichment analysis indicated that the differentially methylated genes (DMGs) were significantly associated with epithelial–mesenchymal transition (EMT) . By developing a novel EMT scoring metric, we found that BrC samples had more of a mesenchymal phenotype than samples from healthy individuals. CDH1, WNT2, and TRIM29 were hypermethylated near the promoter region, while COL5A2 was hypermethylated in the coding region. The DNA methylation and EMT changes were validated through The Cancer Genome Atlas Breast Invasive Carcinoma study, which confirmed that DMGs were associated with gene expression change and that our methylation-based EMT score reliably distinguished tumors from healthy controls. Our findings support the utilization of the NAF cfDNA cfBS methylation profile for noninvasive BrC screening and pave the way for enhanced early detection of this disease.  \nKeywords: breast cancer; DNA methylation; cell-free DNA; nipple aspirate fluid; wholegenome bisulfite sequenci","cbCaijs4IkJWJI4K","https://ap.wps.com/l/cbCaijs4IkJWJI4K","pdf",6697079,"English","# Abstract\n# Introduction","[{\"question\":\"What role does cfDNA play in breast cancer detection?\",\"answer\":\"cfDNA released by malignant tumor cells provides epigenetic information that can indicate cancer status.\"},{\"question\":\"Why is nipple aspirate fluid (NAF) relevant to cfDNA bisulfite sequencing?\",\"answer\":\"NAF is a noninvasive liquid biopsy that contains biomarkers from nearby cancer cells, offering a potentially enriched source for tumor DNA.\"},{\"question\":\"How did the study link methylation patterns to epithelial–mesenchymal transition (EMT)?\",\"answer\":\"Differentially methylated genes were enriched for EMT, and a novel EMT scoring metric showed a more mesenchymal phenotype in breast cancer samples, validated using TCGA.\"}]","Cell-Free DNA Bisulfite Sequencing Reveals Epithelial–Mesenchymal Transition Signatures for Breast Cancer | PDF",1790200238,48]